This revised P01 application will investigate dendritic cell (DC) based cancer immunotherapy strategies targeting a single model tumor associated antigen, carcinoembryonic antigen (CEA). The overall hypothesis to be tested is whether high frequency and durable antigen specific immune responses will have clinical benefits in cancer patients. Three projects are proposed to extend our previous work of active immunization strategies using transduced DC, by focusing on developing and optimizing heterologous priming and boosting vaccination strategies. Project #1 is based on promising data from our phase I trial of vaccination with recombinant fowlpox (CEA-TRICOM) expressing CEA and a triad of co-stimulatory molecules (B7, ICAM-1, and LFA-1) modified DC. Because we are documenting robust T cell responses and early clinical responses using this vaccine strategy, we will continue development in the setting of minimal residual disease by testing the CEA-TRICOM modified DC versus CEA-TRICOM plus GM-CSF in a phase lI study following potentially curative treatment of metastatic colon cancer. Although the fowl pox vector modified DC strategies are promising, we are aware of limitations of the pox vector systems, and propose to develop two alternative vectors to use in future heterologus priming and boosting strategies. Project #2 proposes the generation and development of recombinant alphavirus based vectors, which offer the advantages of having no pre-existing human immune response, efficient transduction of human DC, and high expression of CEA. Project #3 proposes the generation and development of novel second generation recombinant adenoviral vectors that offer advantages such as lower expression of adenovirus protein, minimal induction of DC apoptosis and/or much higher levels of transgene expression. Each of these novel vectors can be combined with the ongoing fowlpox vectors in future heterologous priming and boosting vaccination trials. These three research projects would be supported by three cores: Core A: Administration and regulatory affairs, Core B: Biostatistics and data management, and Core C: Immunologic monitoring and cell processing. Overall, this program project provides a robust environment to test a fundamental hypothesis and important issues in heterologous prime boosting in cancer immune therapy.